Figure 2.

Disruption of cpg-2. (A) Map of the 4.6-kb SphI fragment derived from the cpg-2 disruption construct and used to transform C. parasitica spheroplasts. A 897-bp BamHI/SphI fragment that contained exons 2, 3, 4, and two-thirds of exon 5 was replaced by a 1.4-kb fragment containing the E. coli hph gene and the A. nidulans trpC promoter (19). Probes used for Southern blot analyses are indicated above the map. The restriction endonulease cleavage sites are: B, BamHI; H, HindIII; Sa, SalI; Sp, SphI. (B) Results of Southern blot analyses for the cpg-2 disruptants. Total nucleic acid (10 μg) from the wild-type strain EP155 (lanes 1 and 4) and cpg-2 disruptants G2-37 (lanes 2 and 5) and G2-39 (lanes 3 and 6) was digested with SphI, separated on a 0.8% agarose gel and transferred to a Hybond-N membrane. DNA in lanes 1–3 was probed with a ³²P-labeled 1.3-kb HindIII/SalI DNA fragment, indicated as probe 1 in A. In lanes 4–6, the blot was stripped and reprobed with a ³²P-labeled 584-bp EcoRI/SalI fragment, indicated as probe 2 in A. The sizes of the expected hybridization bands are indicated at the left. (C) Results of Northern hybridization analyses of cpg-2 transcript accumulation in the wild-type strain EP155 (lane 1) and in cpg-2 disruption mutants G2-37 (lane 2) and G2-39 (lane 3). Total RNA (15 μg) was loaded in each lane, separated in a formaldehyde/1.4% agarose gel, and transferred to a Hybond-N membrane. The blot was hybridized with the ³²P-labeled 584-bp EcoRI/SalI fragment (probe 2 in A). The size of the transcript is indicated at the left. After autoradiography, the blot was stripped and rehybridized with a C. parasitica β-tubulin gene cDNA probe (16).